Multiplexed analysis of target nucleic acids

Abstract

The present invention provides, among other things, methods of detecting target nucleic acid, comprising steps of: a) contacting a sample with one or more capturing probes, each comprising at least one target capturing sequence, under conditions that permit the one or more capturing probes to capture one or more target nucleic acids in the sample; b) amplifying the captured one or more target nucleic acids in a reaction mixture comprising a detectable entity such that the amplified one or more target nucleic acids are labeled with the detectable entity; c) incubating amplification product with a plurality of re-capturing probes such that the amplified one or more target nucleic acids are re-captured by the plurality of the re-capturing probes; and d) detecting signal generated by detectable entity associated with the re-captured amplified one or more target nucleic acids, wherein the presence and/or abundance of the detectable signal indicates the presence and/or abundance of the one or more target nucleic acids in the sample.

1 . A method of detecting target nucleic acid, comprising steps of:
 a) contacting a sample with a first set of hydrogel particles bearing one or more capturing probes, each comprising at least one target capturing sequence, under conditions that permit the one or more capturing probes to capture one or more target nucleic acids in the sample;   b) separating the captured target nucleic acid from the first set of hydrogel particles, thereby separating the captured target nucleic acid from the hydrogel particles prior to amplification;   c) amplifying the target nucleic acids in a reaction mixture comprising a detectable entity such that the amplified one or more target nucleic acids are labeled with the detectable entity;   d) incubating amplification product with a second set of hydrogel particles bearing a plurality of re-capturing probes such that the amplified one or more target nucleic acids are recaptured by the plurality of the re-capturing probes, wherein the first set and second set of hydrogel particles are identical, and wherein the re-capturing probes are embedded throughout matrix of the hydrogel particle; and   e) detecting signal generated by detectable entity associated with the re-captured amplified one or more target nucleic acids, wherein the presence and/or abundance of the detectable signal indicates the presence and/or abundance of the one or more target nucleic acids in the sample.   
     
     
         2 . The method of  claim 1 , wherein each of the capturing probes comprises one target capturing sequence and binds specifically to one distinct target nucleic acid or binds to multiple distinct target nucleic acids. 
     
     
         3 . (canceled) 
     
     
         4 . The method of  claim 1  wherein each of the re-capturing probes comprises one target capturing sequence and binds specifically to one distinct target nucleic acid or binds to multiple distinct target nucleic acids. 
     
     
         5 - 12 . (canceled) 
     
     
         13 . The method of  claim 1  wherein the particle further comprises one or more encoding regions and wherein the one or more encoding regions bear detectable moieties that give the identity of the capturing or re-capturing probes. 
     
     
         14 . The method of  claim 1  wherein the one or more target nucleic acids are microRNAs, mRNAs, non-coding transcripts, genomic DNA, cDNAs, siRNAs, DNA/RNA chimera, or combination thereof. 
     
     
         15 . The method of  claim 1  ,wherein the probe is DNA, RNA, DNA/RNA chimera, or combination thereof. 
     
     
         16 . (canceled) 
     
     
         17 . The method of  claim 1  ,wherein the method further comprises a step of coupling one or more adapters to the captured target nucleic acid. 
     
     
         18 - 21 . (canceled) 
     
     
         22 . The method of  claim 17  , wherein each of the capturing probes further comprises sequences complementary to the one or more adapters. 
     
     
         23 . The method of  claim 22 , wherein the sequences complementary to the one or more adapters are adjacent to the target capture sequence. 
     
     
         24 - 26 . (canceled) 
     
     
         27 . The method of  claim 1  ,wherein the step of amplifying the captured target nucleic acid comprises performing a polymerase chain reaction (PCR). 
     
     
         28 - 36 . (canceled) 
     
     
         37 . The method of  claim 27  , wherein the PCR is performed with primers attached to a substrate. 
     
     
         38 - 42 . (canceled) 
     
     
         43 . The method of  claim 37  ,wherein the substrate is present during the time of amplification. 
     
     
         44 - 55 . (canceled) 
     
     
         56 . The method of  claim 1  ,wherein the signal generated by detectable entity is detected by a flow cytometer, or array scanner. 
     
     
         57 - 66 . (canceled) 
     
     
         67 . The method of  claim 1  , wherein each target nucleic acid represents less than 1% of total nucleic acids in the biological sample. 
     
     
         68 - 70 . (canceled) 
     
     
         71 . A method of detecting target nucleic acid, comprising steps of:
 a) contacting a sample comprising one or more target nucleic acids with a first set of hydrogel particles bearing a plurality of capturing probes, each comprising at least one target capturing sequence, under conditions that permit the plurality of capturing probes to capture one or more target nucleic acids in the sample;   b) separating the captured target nucleic acid from the first set of hydrogel particles, thereby separating the captured target nucleic acid from the hydrogel particles prior to amplification;   c) amplifying the captured one or more target nucleic acids in a reaction mixture comprising a detectable entity such that the amplified one or more target nucleic acids are labeled with the detectable entity; and   d) incubating amplification product with a second set of hydrogel particles bearing a plurality of re-capturing probes such that the amplified one or more target nucleic acids are re-captured by the plurality of the re-capturing probes, wherein the first and the second set of hydrogel particles are identical, and wherein the re-capturing probes are embedded throughout matrix of the hydrogel particle; 
 wherein each particle has one or more encoding regions bearing detectable moieties that give the identity of the capturing or re-capturing probes thereon; and 
 wherein the presence and/or abundance of the detectable signal generated by detectable entity associated with the re-captured amplified one or more target nucleic acids on the second set of particles indicates the presence and/or abundance of the one or more target nucleic acids in the sample. 
   
     
     
         72 - 144 . (canceled) 
     
     
         145 . A kit for detecting target nucleic acid, comprising:
 particles comprising one or more probe regions bearing probes and one or more encoding regions bearing detectable moieties that give the identity of the probes thereon, wherein the probes comprise target capturing sequence;   a hybridization buffer with pre-determined ionic strength, buffered pH, and denaturing reagent;   a labeling buffer comprising adapters designed to serve as sites for polymerase chain reaction priming and/or reverse transcription; and   a PCR buffer containing primers, dNTPs, and reagents for amplification of captured targets.   
     
     
         146 - 149 . (canceled)